Effect of sequential delivery of 1‐ and 2‐MHz bipolar microneedling radiofrequency energy on thermal tissue reactions in a minipig model

Abstract Background Bipolar microneedling radiofrequency (RF) treatment generates different patterns of thermal reactions, depending on the skin impedance and RF treatment parameters, including the frequency, power, conduction time, settings of sub‐pulse packs, and penetrating depth and type of microneedles used. We compared the effect of sequential delivery of 1‐ and 2‐MHz bipolar RF energy to in vivo minipig skin on thermal tissue reaction. Methods RF treatments at frequencies of 1 and 2 MHz were sequentially delivered to minipigs’ skin in vivo. A histological study was performed to analyze RF‐induced skin reactions at 1‐h and at 3‐, 7‐, and 14‐days post‐treatment. Results The skin specimens demonstrated that the two different frequencies of RF treatment generated mixed patterns of the peri‐electrode coagulative necrosis (PECN) according to the experimental settings and tissue impedance. In the PECN zone, the tissue coagulation induced by the first RF treatment was surrounded by the effect of the later RF treatment at the other RF frequency. In the inter‐electrode non‐necrotic thermal reaction zone, the effect of the latter RF treatment was widespread and deep through the dermis, which had received RF treatment at the other frequency first. The delivery of pulsed‐type RF energy at sub‐pulse packs of 6 or 10 provided effective RF delivery over long conduction time without excessive thermal damage of the epidermis. Nonetheless, by sequential delivery of two different RF frequencies, RF‐induced tissue reactions were found to be markedly enhanced. Conclusion The sequential delivery of 1‐ and 2‐MHz RF energy induces novel histological patterns of tissue reactions, which can synergistically enhance the thermostimulatory effects of each RF setting. Moreover, variations in patterns of tissue reactions can be generated by regulating the order of frequencies and the number of sub‐pulse packs of RF used.


INTRODUCTION
Bipolar, alternating current, microneedling radiofrequency (RF) treatment induces thermal reactions in the targeted areas of the skin.][6][7][8][9][10][11] Recently, the effects of sub-pulse packs on RF-induced tissue reactions have been reported. 3,5,12Various factors, including the number of sub-pulse packs, on-time of the sub-pulse pack, off-time between sub-pulse packs, and total treatment time, which refers to the sum of total RF conduction time and total off-time, can be regarded as significant variations during pulsed-type RF treatment. 3,124][5] Coagulation necrosis in the perielectrode area can be generated from the tips of microneedles when bipolar alternating current RF energy is delivered to areas of homogeneous tissue impedance. 6However, the location of generating a necrotic thermal reaction may differ along the peri-electrode area in the layered tissues with abundant adnexal components presenting various levels of tissue impedance. 3,4With the assumption that no desiccative tissue reactions occur in the peri-electrode area, which would inhibit further propagation of RF pulses, non-necrotic thermal tissue reactions are found in the inter-electrode area, without marked tissue necrosis, as observed by microscopy. 31][12] In this observational and descriptive study, we compared the effects of sequentially delivered 1-and 2-MHz RF energy on RF-induced early and late thermal reactions of the skin, using a bipolar, alternating current, microneedling RF device on minipig skin in vivo.Once the microneedles were inserted into the minipig's skin, RF treatment at two different frequencies (1 and 2 MHz) were sequentially delivered at 50-ms intervals.Additionally, RF treatment at each frequency was delivered under various conditions, by regulating the number of sub-pulse packs, total treatment time, RF conduction time, and total off-time.Minipig skin specimens, which were obtained at 1-h and 3-, 7-, and 14-days after RF treatment, were then microscopically analyzed.General anesthesia was administered via an intramuscular bolus injection of tiletamine/zolazepam (5 mg/kg) and xylazine (2 mg/kg).

In vivo minipig model
Thereafter, we performed endotracheal intubation and connected the pig to a ventilator.The lungs were ventilated with oxygen, and anesthesia was maintained with 2% isoflurane.Intravenous hydration with normal saline was maintained through a superficial auricular vein (25 mL/h).After gentle removal of the hairs on the abdomen, the lesions were cleansed with a mild soap and 70% alcohol.The skin was marked using black ink to outline a grid for each experimental setting (15 mm × 15 mm/grid; 40 grids/minipig; total of 160 grids).

Bipolar RF device and experimental parameters
An invasive bipolar, alternating current, pulsed type, microneedling RF device (VIRTUE RF™; Shenb Co., Ltd., Seoul, Korea) was used at frequencies of 1 and 2 MHz to analyze RF tissue reactions in in vivo porcine skin.The device delivered RF energy through 36 insulated microneedle electrodes (24 K gold-plated surgical stainless steel) in a 15-mm × 15-mm disposable tip (Deep RF™ 36 tip; Shenb Co., Ltd.).
The electrodes within this tip were uniformly arranged in a 6 × 6 pattern, with the nearest distance between microneedles being 3 mm.Parallel contact cooling (PCC), using a cooled sapphire metal plate with thermoelectric elements, was adapted to protect the epidermis from thermal injury.This plate was integrated into the handpiece.The temperature of the chilled plate was regulated as 15 • C± 0.5 • C for this experiment.
In this study, RF treatment at frequencies of 1 and 2 MHz was performed on each grid of the experimental minipigs at a power of 35 W and an electrode penetration depth of 1.5 mm in one pass.Once the microneedles were inserted into the minipig's skin, RF treatment F I G U R E 1 Schematic illustrations of experimental parameters for sequential invasive delivery of 1-and 2-MHz bipolar radiofrequency (RF) energy on in vivo minipig skin tissue.Experimental settings of the frequency of 1 MHz, RF conduction time of (A) 200, (B) 300, and (C) 500 ms, and a single pulse pack (1-200/300/500-1) and additional RF treatment at 2 MHz and the single pulse pack of corresponding RF conduction time settings (2-200/300/500-1) with the interval settings between the two types of RF frequencies at 50 ms.Likewise, 2-MHz RF treatment was followed by 1-MHz RF treatment at the settings of (D) 2-200-1 + 1-200-1, (E) 2-300-1 + 1-300-1, and (F) 2-300-1 + 1-300-1.
Secondly, RF treatment at 1 MHz, for a total treatment time of 500 ms (total RF conduction time, 250 ms and total off-time, 250 ms) and 1000 ms (total RF conduction time, 500 ms and total off-time, 500 ms) with six sub-pulse packs (1-500/1000-6), and additional RF pulses at 2 MHz, for a total treatment time of 500 and 1000 ms with six sub-pulse packs (2-500/1000-6) were sequentially delivered at 50-ms intervals (Figure 2A-F).The total treatment time referred to the sum of the total RF conduction time and the total off-time.Likewise, RF treatment at 2-500/1000-6 and additional RF pulses at 1-500/1000-6 were sequentially delivered at 50-ms intervals.Additionally, RF treatment at 1-1200-10 (total RF conduction time, 120 ms, and total off-time, 1080 ms) and additional RF pulses at 2-1200-10 were sequentially delivered at 50-ms intervals.Similarly, RF pulses at 2-1200-10 and additional RF treatment at 1-1200-10 were sequentially delivered at 50-ms intervals.
delivered at 50-ms intervals.RF treatment at 2-500-6 and additional RF treatment at 1-500-1 were also sequentially delivered at 50-ms intervals.Neither pretreatment topical anesthesia nor pre-and postcooling was applied.No prophylactic topical or systemic antibiotics or steroids were used after RF treatment.

Histological evaluation
The experimental minipigs were euthanized to sample the treated tissue in a humane manner, according to standard protocols.One hour and 3, 7, and 14 days after treatment, full-thickness tissue specimens, including the epidermis, dermis, and subcutaneous fat were obtained for microscopic evaluation.Each sample was fixed in 10% buffered formalin and was embedded in paraffin.Then, serial tissue sections of 4-µm thickness for each treatment setting were prepared and were stained with hematoxylin and eosin.
Moreover, RF treatment at 2-500-6 + 1-500-6 generated PECN areas in the upper dermis, which were notably larger and deeper at settings of 2-1000-6 + 1-1000-6 and 2-1200-10 + 1-1200-10, and to that in other treatment settings with multiple sub-pulse packs (Figure 8A-D).Notable histological features of collagen regeneration in the IENT zones were found under all experimental conditions.On day 14, sequential 1-MHz RF delivery followed by 2-MHz RF generated diffuse areas of neocollagenesis in the entire dermis, and the degree of which seemed to be more remarkable depending on the overall RF treatment time.Moreover, sequential 2-MHz RF delivery followed by 1-MHz RF treatment resulted in 2-MHz, multiple sub-pulse pack-induced polymorphous areas of neocollagenesis on day 14.These tissue reactions appeared to be enhanced by subsequent 1-MHz RF delivery (Figure 8E-H).

Effect of sequential 1-and 2-MHz, single pulse, and multiple sub-pulse pack RF delivery
The minipig skin specimen, which was obtained after RF treatment at a setting of 1-500-1 + 2-500-6, exhibited 1-MHz RF-induced, predominantly basophilic round to oval PECN lesions with 2-MHz RF-induced, fractionated eosinophilic coagulation of the collagen bundles.In these lesions, notable RF-induced, diffusely edematous thermal reactions with eosinophilic tissue reactions were also found in the mid to deep dermis between the microneedles.Furthermore, RF treatment at a setting of 1-500-6 + 2-500-1 generated noticeable, but smaller PECN areas than did that at a setting of 1-500-1 + 2-500-6.Nonetheless, 2-MHz RF-induced, necrotic thermal reactions, which surrounded the 1-MHz RF-induced, basophilic PECN areas, produced high-degree thermal tissue reactions that were more apparent in the specimens at 3-days post-RF (Figure 9A-D).Moreover, RF-induced, multiple, small eosinophilic patches of high-degree, but non-coagulative, thermal reactions, were found in the upper, mid, and deep dermis around and between the microneedle insertions.
In these regions, thermal reactions in the dermis seemed to be less marked than those obtained with other experimental settings.

DISCUSSION
In this experimental pilot study, we histologically evaluated the effects In previous studies, RF treatment at a frequency of 0.5 or 1 MHz generated PECN zone with water drop-or chandelier-like appearance in the upper or mid dermis, depending on the impedance of the target tissue. 10,11,15RF treatment at 2 MHz resulted in narrower, but more intensely injured PECN areas than did RF at other frequency settings. 10,11,15Moreover, RF treatment at a frequency of 0.5 or 1 MHz generated diffuse, edematous IENT areas in the dermis, whereas 2-MHz RF treatment demonstrated notable thermal changes of the collagen bundles around the microneedles after a shorter conduction time, which propagated toward the paired microneedles with increasing conduction time. 10,15 the present study, we found that sequential delivery of two dif- We propose that those histological changes could have resulted from the cumulative RF-induced thermal effects due to upward heat diffusion with enhanced impedance changes over a long treatment period.
In this study, we used PCC during RF treatment for all experimental settings to prevent excessive thermal injury to the epidermis and upper papillary dermis. 13,14Skin cooling during energy-delivering treatments has been adapted to reduce patients' pain and post-treatment adverse events. 13,14Among the methods of skin cooling, including contact/non-contact cooling and pre-cooling/parallel cooling/postcooling, we opted to use PCC during RF treatments. 12Our research group has previously demonstrated the effects of PCC on RF-induced thermal reactions of in vivo minipig skin by using thermometric and histological analyses. 15According to our previous study, PCC effectively protects the epidermis and upper papillary dermis against excessive thermal tissue injury, as compared to RF treatment without PCC. 15rthermore, PCC markedly enhanced RF-induced skin reactions in the mid to deep dermis at both early and late stages of wound healing and tissue regeneration. 15[6]11 The number of experimental RF parameters adjusted when delivering the treatment at two different frequencies were limited in this pilot study and the effects of RF treatment on the skin were only histologically evaluated in this study.Moreover, the amount of collagen fibers and appendages between minipig and human skin differ, which limits the extrapolation of our data to clinical situations for treating various dermatological conditions.Although our data exhibited no desiccative tissue reactions in the epidermis and dermis of the RF-treated minipig skin specimens, high power and long RF treatment time settings were selected in our experiment to obtain histologically distinguishable differentiating points among RF settings.Therefore, when sequentially delivering two different RF frequencies to human skin, treatment parameters will need to be adjusted, depending on the skin reactions, to ensure safety and efficacy.Additionally, our data could not demonstrate representative thermal tissue reactions to treatment with all bipolar, alternating current, pulsed-type RF devices.We consider that different patterns of RF-induced tissue reactions could be obtained according to the type of device used, which have subtle technological differences.

CONCLUSION
In this study, we evaluated the effect of sequential delivery of 1-and 2-MHz RF energy on early and late thermal tissue reactions in in vivo minipig skin using a bipolar microneedling RF device.Our data revealed that sequential delivery of RF treatment to the skin at two frequencies generated mixed patterns of the PECN according to the experimental settings and tissue impedance.In the PECN zone, the tissue coagulation induced by the initial RF treatment at one frequency was found to be surrounded by the thermal effect of the subsequent RF treatment at the other RF frequency.In the IENT zone, the effects of the second RF treatment were widely and deeply spread through the dermis after the first RF treatment at the other frequency.However, additional research using mixed RF treatment settings involving different frequencies and sub-pulse packs are needed to confirm our findings.Moreover, controlled clinical studies are required before using sequential 1-and 2-MHz RF delivery to human skin in clinical applications.

Four 10 -
week-old female minipigs (Sus scrofa domestica) were purchased (M-pig ® ; CRONEX Inc., Cheongju, Korea), and the in vivo experiments were performed at the age of 12 weeks, when the pigs weighted 55−75 kg.Ethical approval for this study was obtained from the Ethics Committee of the Institutional Animal Care and Use Committee in CRONEX Inc. in Cheongju, Korea (CRONEX-IACUC: 202210005).All animal experiments were performed in accordance with the guidelines of the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and the Institutional Animal Care and Use Committee.

of 1 - 10 F I G U R E 9
and 2-MHz RF energy sequentially delivered to the skin using a bipolar, alternating current, microneedling RF device, under various experimental settings differing in terms of the number of sub-pulse packs, total treatment time, RF conduction time, and total off-time for each frequency.The two different frequencies generated mixed patterns of PECN, varying with the experimental settings and tissue impedance.In the PECN zone, the tissue coagulation induced by the first RF treatment at one frequency was surrounded by the effect of the later RF treatment at the other RF frequency.In the IENT zone, the effect of the latter RF treatment was widespread and deep through the dermis that had undergone initial RF treatment at the other frequency.The delivery of pulsed-type RF energy at sub-pulse packs of 6 or Immediate and early tissue reactions in in vivo minipig skin after sequential 1-and 2-MHz RF delivery at various sub-pulse pack settings.The skin of a minipig was treated with RF at the settings of (A, B) 1-500-1 + 2-500-6, (C, D) 1-500-6 + 2-500-1, (E, F) 2-500-1 + 1-500-6, and (G, H) 2-500-6 + 1-500-1.The skin specimens were obtained (A, C, E, G) immediately and (B, D, F, H) 3 days after RF treatment.Asterisks, peri-electrode coagulative necrosis zones.Hematoxylin and eosin staining.Original magnification × 40.provided effective RF delivery over long conduction time, without causing excessive thermal damage in the epidermis.Nonetheless, sequential delivery of two different RF frequencies resulted in more marked RF-induced tissue reactions.
ferent RF frequencies generated mixed patterns of PECN depending on the experimental settings and tissue impedance.The histological patterns of the PECN seemed to be composed of earlier RF-induced tissue necrosis surrounded by later RF-induced thermal reactions.When the former RF frequency was set to 1 MHz, round to oval, relatively basophilic PECN zones were found in the dermis, with a size larger than the PECN zones induced by 2 MHz RF.When the initial RF treatment was set to 2 MHz, oval to cylindrical, relatively eosinophilic PECN zones were formed in the dermis, and the degree of tissue degeneration seemed to be greater than that observed after 1 MHz treatment.Moreover, the histological features suggested that some PECN lesions in the upper to mid dermis, which corresponded to the level of the insulated part of the microneedles, could have resulted from accumulated thermal reactions induced by RF flow in the upper dermis with its high tissue impedance.A gated delivery of RF energy reportedly generates wider ranges of non-necrotic dermal tissue reactions with a lesser degree of coagulative thermal reactions and is achieved by increasing the number of sub-pulse packs.3,5In this study, although the settings of total treatment time (500 ms) and six sub-pulse packs delivered only 250 ms of RF energy.The experimental conditions of 1-500-1 + 2-500-6 notably presented 2-MHz RF-induced IENT throughout the dermal layers.We suggested that single-pulse, 1-MHz RF delivery initially generated the PECN and the widely distributed IENT in the dermis.Then, subsequent 2-MHz RF treatment at multiple sub-pulse-settings generated lesser PECN and more marked IENT, along with the changes already present in the 1-MHz pretreated dermal tissues.Furthermore, RF treatment at 2-500-1 + 1-500-6 generated cylindrically coagulated PECN with diffuse and homogenous IENT areas in the dermis.Interestingly, the experimental settings of total treatment time of 1200 ms and the number of sub-pulse packs of 10, which delivered 120-ms RF energy over 1200 ms, some specimen sections showed large areas of PECN with extensive thermal tissue reaction than did other experimental settings.